DESCRIPTION: It is now clear that the brain and immune tissues have a cannabinoid system of receptors and ligands. However, it is not clear how this system is structured and how it affects human physiology and health. This is now a critical issue because the medicinal use of marijuana is gaining political and public support and is now recommended for use in AIDS wasting disease. The overall goal of our research is to understand the immunological effects of cannabimimetics and the natural function of the "immunocannabinoid" system. This proposal is a continuation of a project started in 1998 on defining the cannabinoid receptor (CBR) mRNA expression in murine immune cells. We have been able to show that immune subsets from mouse and human differ in their expression profile ranging from no expression to high expression. Furthermore, activated immune subsets modulate CBR mRNA expression. The present application is an extension of these findings. In Aim 1, we will define the distribution profile of CB1 and CB2, both mRNA and protein, in T cells, B cells, NK cells, and macrophages from mouse tissues and human PBMCs. It is our hypothesis that immune subsets have different basal levels of CBR expression commensurate with the putative role of these receptors in the immunobiology of the cell. Purified subsets will be analyzed by RT-PCR, flow cytometry, Western blotting, immunoprecipitation, and biotin labeling of surface proteins. We expect to document the CBR phenotype of key immune tissues. In Aim 2, we will define the changes in the CBR profile following cell activation and possible mechanisms of these changes. It is our hypothesis that CBR gene activity and receptor expression are regulated by the gene activation sequence in immune cells. Subsets will be mitogen and cytokine activated and analyzed for receptor expression by protein analysis and real-time quantitative PCR. Because there are at least 5 different mRNAs associated with CB1, variations in exon usage in different immune subsets will be analyzed by transcription analysis, mRNA usage by RT-PCR and RNase protection, and 5' end mapping with 5' RACE. We expect to document changes in the CBR expression profile in activated subsets and to discover the CB1 exon usage pattern. In Aim3, we will define the putative role of the cannabinoid system in cell proliferation, TNF production, and IL-12-induced Th1 activity. It is our hypothesis that these functions are regulated by the "immunocannabinoid" sytem. The experiments proposed will increase our understanding of the imnunobiology of the cannabinoid system and shed light on the health consequences of medicinal marijuana use in HIV disease.